The proposed work is aimed at elucidating the mode of formation of 2,6-dideoxyhexoses, commonly found as sugar moieties of antibiotics, from their precursor, glucose. Three systems will be studies: (a) the 2-deoxyrhamnose moieties found in chlorothricin, produced by Streptomyces antibioticus, (b) the 2,6-dideoxy-ketohexose-derived sugar moiety of granaticin, a metabolite of S. violaceoruber and (c) the L-daunosamine moiety of the antitumor antibiotic daunomycin from S. peucetius. The pathways to these sugars will be established by incubating cell-free extracts of the producing organisms with radiolabeled TDP-4-keto-6-deoxy- glucose, the established first pathway-specific intermediate of deoxyhexose biosynthesis in Streptomycetes, examining the incubations for conversion products and determining their structures. This will be followed by isolation, purification and characterization of the enzymes catalyzing individual steps in this sequence, and their mechanistic analysis. In a parallel approach we will attempt to clone the genes coding for these enzymes and to express them in a heterologous host or to overexpress them in the parent organism. Two probes will be used to search for these genes: (a) an oligonucleotide probe derived from the amino acid sequence of the TDP-glucose oxidoreductase purified from the parent organism and (b) genes involved in the formation of the polyketide moieties of the parent antibiotics. Our approach is predicated on the assumption that the biosynthetic genes, as with other Streptomyces antibiotics, will be arranged in clusters. Further genetic analysis may reveal how these genes are regulated and how the production of the alycones and the sugar moieties are correlated.